Ursolic acid morpholine and diethanolamine salts

ABSTRACT

The invention provides diethanolamine and morpholine salts of ursolic acid. Compositions containing the salts, and methods of using the salts are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalApplication No. PCT/US2018/049742, filed Sep. 6, 2018, and published asWO 2019/055280 on Mar. 21, 2019, which claims the benefit of U.S.Provisional Application Nos. 62/558,004 (filed Sep. 13, 2017) and62/649,938 (filed Mar. 29, 2018). The entire contents of each of theprior applications are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides novel diethanolamine and morpholine saltsof ursolic acid, which exhibit superior efficacy and properties.Compositions comprising the salts, and methods employing the salts arealso provided.

BACKGROUND OF THE INVENTION

Ursolic acid is a pentacyclic triterpene acid. A range of biologicaleffects of ursolic acid are discussed in WO 2011/146768 and WO2012/170546. At the molecular level, ursolic acid inhibits the STAT3activation pathway, reduces matrix metalloproteinase-9 expression viathe glucocorticoid receptor, inhibits protein tyrosine phosphatases,acts as an insulin mimetic, activates PPARα, inhibits NF-kBtranscription factors, translocates hormone-sensitive lipase tostimulate lipolysis and inhibits the hepatic polyol pathway, among othereffects.

SUMMARY OF THE INVENTION

Briefly, the present invention provides novel ursolic aciddiethanolamine and morpholine salts, as well as related compositions andmethods. The salts exhibit superior properties, including effects onskeletal muscle, as compared to ursolic acid per se and other ursolicacid salts.

Certain embodiments of the presently-disclosed ursolic acid salts,compositions comprising the salts, and methods employing the salts haveseveral features, no single one of which is solely responsible for theirdesirable attributes. Without limiting the scope of the ursolic acidsalts, compositions comprising the salts, and methods employing thesalts as defined by the claims that follow, their more prominentfeatures will now be discussed briefly. After considering thisdiscussion, and particularly after reading the section of thisspecification entitled “Detailed Description of the Invention,” one willunderstand how the features of the various embodiments disclosed hereinprovide a number of advantages over the current state of the art. Forexample, embodiments of the invention provide unexpectedly superiorproperties compared to, e.g., native ursolic acid and other ursolic acidsalts, including, e.g., efficaciousness in stimulating skeletal musclehypertrophy, increasing lean muscle mass, decreasing fat mass,increasing skeletal muscle fiber size, decreasing adipocyte size,reducing obesity and/or blood glucose, improving glucose tolerance, etc.

In a first aspect, the invention provides an ursolic acid salt selectedfrom ursolic acid diethanolamine salt and ursolic acid morpholine salt.

In a second aspect, the invention provides a composition comprising anursolic acid salt selected from ursolic acid diethanolamine salt andursolic acid morpholine salt, and a further agent.

In a third aspect, the invention provides a method for:

-   -   (i) increasing skeletal muscle mass;    -   (ii) treating skeletal muscle atrophy;    -   (iii) treating sarcopenia;    -   (iv) treating cachexia;    -   (v) increasing strength;    -   (vi) treating weakness;    -   (vii) increasing exercise capacity;    -   (viii) treating fatigue;    -   (ix) promoting muscle growth;    -   (x) promoting normal muscle function;    -   (xi) improving muscle function;    -   (xii) promoting muscle health;    -   (xiii) promoting healthy aging in muscles;    -   (xiv) increasing energy expenditure;    -   (xv) increasing the ratio of skeletal muscle to fat;    -   (xvi) reducing fat;    -   (xvii) treating obesity;    -   (xviii) treating diabetes;    -   (xix) lowering blood glucose;    -   (xx) treating pre-diabetes;    -   (xxi) treating metabolic syndrome;    -   (xxii) treating insulin resistance;    -   (xxiii) reducing plasma cholesterol;    -   (xxiv) treating hypercholesterolemia;    -   (xxv) reducing plasma triglycerides;    -   (xxvi) treating hypertriglyceridemia;    -   (xxvii) promoting healthy metabolism;    -   (xxviii) promoting metabolic health;    -   (xxix) treating hypertension;    -   (xxx) treating atherosclerosis    -   (xxxi) treating myocardial ischemia;    -   (xxxii) treating myocardial infarction;    -   (xxxiii) treating cardiomyopathy;    -   (xxxiv) treating cardiac arrhythmia;    -   (xxxv) treating non-alcoholic fatty liver disease (NAFLD);    -   (xxxvi) treating liver fibrosis;    -   (xxxvii) treating liver injury;    -   (xxxviii) treating lung injury;    -   (xxxix) treating gastric ulcers;    -   (xl) treating nephropathy;    -   (xli) promoting bone formation;    -   (xlii) promoting normal bone structure;    -   (xliii) promoting bone health;    -   (xliv) treating osteoporosis;    -   (xlv) treating cerebral ischemia;    -   (xlvi) treating cerebral hemorrhage;    -   (xlvii) treating stroke;    -   (xlviii) treating traumatic brain injury;    -   (xlix) treating dementia;    -   (l) treating Alzheimer's disease;    -   (li) treating memory loss;    -   (lii) treating cognitive dysfunction;    -   (liii) promoting normal cognitive function;    -   (liv) treating anxiety;    -   (lv) treating depression;    -   (lvi) reducing inflammation;    -   (lvii) treating arthritis;    -   (lviii) treating skin ulcers;    -   (lix) treating skin wounds;    -   (lx) promoting wound healing;    -   (lxi) treating skin dryness;    -   (lxii) treating skin roughness;    -   (lxiii) treating skin scarring;    -   (lxiv) treating skin wrinkles;    -   (lxv) reducing unwanted effects of aging;    -   (lxvi) treating cancer;    -   (lxvii) reducing tumor growth;    -   (lxviii) treating tumor metastasis;    -   (lxix) treating tumor angiogenesis;    -   (lxx) increasing tumor cell apoptosis;    -   (lxxi) decreasing protein oxidation;    -   (lxxii) decreasing lipid oxidation;    -   (lxxiii) decreasing DNA oxidation;    -   (lxxiv) decreasing RNA oxidation;    -   (lxxv) decreasing oxidation of cellular molecules;    -   (lxxvi) decreasing DNA damage;    -   (lxxvii) treating bacterial infection;    -   (lxxviii) reducing bacterial growth;    -   (lxxix) treating fungal infection;    -   (lxxx) reducing fungal growth;    -   (lxxxi) treating viral infection;    -   (lxxxii) treating protozoal infection;    -   (lxxxiii) treating nematode infection; or    -   (lxxxiv) treating a disease state, condition, or disorder        mediated by activating transcription factor 4 (ATF4),        in a subject, the method comprising administering to the subject        an ursolic acid salt selected from ursolic acid diethanolamine        salt and ursolic acid morpholine salt, or a composition        comprising the ursolic acid salt.

These and other features and advantages of this invention will becomeapparent from the following detailed description of the various aspectsof the invention taken in conjunction with the appended claims and theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will hereinafter be described in conjunction withthe following drawing figures, and:

FIGS. 1A and 1B are photomicrographs of quadriceps skeletal musclefibers from control and test mice, respectively, after 7 weeks of adlibitum access to either standard chow (control) or standard chowsupplemented with 0.059% ursolic acid-morpholine salt.

FIGS. 2A and 2B are photomicrographs of adipocytes from retroperitonealfat pads from control and test mice, respectively, after 7 weeks of adlibitum access to either standard chow (control) or standard chowsupplemented with 0.059% ursolic acid-morpholine salt.

FIG. 3 is a chart depicting glucose tolerance testing results fromcontrol and test mice after 8 weeks of ad libitum access to eitherhigh-fat chow (control) or high-fat chow supplemented with 0.059%ursolic acid-morpholine salt.

FIG. 4 is a graph depicting glucose tolerance testing results.

FIG. 5 is a graph depicting glucose tolerance testing results.

FIG. 6 is a graph depicting testing results for percent change in totalcellular protein.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention and certain features, advantages, anddetails thereof, are explained more fully below. Descriptions ofwell-known materials, equipment, processing techniques, etc., areomitted so as to not unnecessarily obscure the invention in detail.

In a first aspect, the invention provides an ursolic acid salt selectedfrom ursolic acid diethanolamine salt and ursolic acid morpholine salt.

The inventive ursolic acid salts can be synthesized by techniques knownin the art. The starting materials of the compounds of this inventionare available from commercial sources or can themselves be synthesizedusing reagents and techniques known in the art. Non-limiting synthesisembodiments are described herein.

Ursolic acid, which has the formula

is commercially available, and is present in various plants from whichit can be extracted, including apples, basil, bilberries, cranberries,elder flower, peppermint, rosemary, lavender, oregano, thyme, hawthorn,and prunes.

In some embodiments, the inventive ursolic acid salt is ursolic aciddiethanolamine salt of formula

In some embodiments, the inventive ursolic acid salt is ursolic acidmorpholine salt of formula

In a second aspect, the invention provides a composition comprising anursolic acid salt selected from ursolic acid diethanolamine salt andursolic acid morpholine salt, and a further agent.

The further agent is a component, in addition to the ursolic acid salt,that is present in the composition. Persons having ordinary skill in theart are familiar with various types of further agents that may beincluded in a composition. For example, in some non-limitingembodiments, the further agent comprises an excipient, binder, diluent,carrier, other delivery vehicle or system, filler, salt, buffer,preservative, antioxidant, stabilizer, sweetening agent, or flavoragent.

In some embodiments, the further agent is a physiologically acceptableagent. As used herein, a physiologically acceptable agent is an agentthat does not show significant toxicity to an intended subject at thedose of administration. The term physiologically acceptable agentcomprises generally regarded as safe (GRAS) substances. GRAS substancesare listed by the Food and Drug Administration in the Code of FederalRegulations (CFR) at 21 CFR. § 182 and 21 CFR § 184. The August 2017versions of 21 CFR. § 182 and 21 CFR § 184 are hereby incorporatedherein by reference. The term physiologically acceptable agent alsocomprises pharmaceutically acceptable agents.

The term “pharmaceutically acceptable,” as used herein, refers to acomponent that is, within the scope of sound medical judgment, suitablefor use in contact with the tissues of a subject (e.g., human or otheranimal) without undue toxicity, irritation, allergic response and thelike, and is commensurate with a reasonable benefit/risk ratio.

In some embodiments, the inventive composition is formulated forpharmaceutical use (i.e., is “a pharmaceutical composition”).Pharmaceutical compositions comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle that is “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof in amounts typically used inmedicaments.

Pharmaceutically acceptable carriers, adjuvants and vehicles that can beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

In some embodiments, the composition is a pharmaceutical composition,nutraceutical composition, food composition, or dietary supplement.

In some embodiments, the composition is in the form of, or comprises anon-naturally occurring oral delivery vehicle. Such delivery vehiclesexclude naturally occurring vehicles such as plants and fruits. Oraldelivery vehicles include, but are not limited to capsules, pills,tablets, cachets, syrups, foods, and beverages.

In some embodiments, the composition is in the form of one or moretablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to asubject, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil-in-water emulsions, or a water-in-oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a patient; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a subject.

In a third aspect, the invention provides a method for:

-   -   (i) increasing skeletal muscle mass;    -   (ii) treating skeletal muscle atrophy;    -   (iii) treating sarcopenia;    -   (iv) treating cachexia;    -   (v) increasing strength;    -   (vi) treating weakness;    -   (vii) increasing exercise capacity;    -   (viii) treating fatigue;    -   (ix) promoting muscle growth;    -   (x) promoting normal muscle function;    -   (xi) improving muscle function;    -   (xii) promoting muscle health;    -   (xiii) promoting healthy aging in muscles;    -   (xiv) increasing energy expenditure;    -   (xv) increasing the ratio of skeletal muscle to fat;    -   (xvi) reducing fat;    -   (xvii) treating obesity;    -   (xviii) treating diabetes;    -   (xix) lowering blood glucose;    -   (xx) treating pre-diabetes;    -   (xxi) treating metabolic syndrome;    -   (xxii) treating insulin resistance;    -   (xxiii) reducing plasma cholesterol;    -   (xxiv) treating hypercholesterolemia;    -   (xxv) reducing plasma triglycerides;    -   (xxvi) treating hypertriglyceridemia;    -   (xxvii) promoting healthy metabolism;    -   (xxviii) promoting metabolic health;    -   (xxix) treating hypertension;    -   (xxx) treating atherosclerosis    -   (xxxi) treating myocardial ischemia;    -   (xxxii) treating myocardial infarction;    -   (xxxiii) treating cardiomyopathy;    -   (xxxiv) treating cardiac arrhythmia;    -   (xxxv) treating non-alcoholic fatty liver disease (NAFLD);    -   (xxxvi) treating liver fibrosis;    -   (xxxvii) treating liver injury;    -   (xxxviii) treating lung injury;    -   (xxxix) treating gastric ulcers;    -   (xl) treating nephropathy;    -   (xli) promoting bone formation;    -   (xlii) promoting normal bone structure;    -   (xliii) promoting bone health;    -   (xliv) treating osteoporosis;    -   (xlv) treating cerebral ischemia;    -   (xlvi) treating cerebral hemorrhage;    -   (xlvii) treating stroke;    -   (xlviii) treating traumatic brain injury;    -   (xlix) treating dementia;    -   (l) treating Alzheimer's disease;    -   (li) treating memory loss;    -   (lii) treating cognitive dysfunction;    -   (liii) promoting normal cognitive function;    -   (liv) treating anxiety;    -   (lv) treating depression;    -   (lvi) reducing inflammation;    -   (lvii) treating arthritis;    -   (lviii) treating skin ulcers;    -   (lix) treating skin wounds;    -   (lx) promoting wound healing;    -   (lxi) treating skin dryness;    -   (lxii) treating skin roughness;    -   (lxiii) treating skin scarring;    -   (lxiv) treating skin wrinkles;    -   (lxv) reducing unwanted effects of aging;    -   (lxvi) treating cancer;    -   (lxvii) reducing tumor growth;    -   (lxviii) treating tumor metastasis;    -   (lxix) treating tumor angiogenesis;    -   (lxx) increasing tumor cell apoptosis;    -   (lxxi) decreasing protein oxidation;    -   (lxxii) decreasing lipid oxidation;    -   (lxxiii) decreasing DNA oxidation;    -   (lxxiv) decreasing RNA oxidation;    -   (lxxv) decreasing oxidation of cellular molecules;    -   (lxxvi) decreasing DNA damage;    -   (lxxvii) treating bacterial infection;    -   (lxxviii) reducing bacterial growth;    -   (lxxix) treating fungal infection;    -   (lxxx) reducing fungal growth;    -   (lxxxi) treating viral infection;    -   (lxxxii) treating protozoal infection;    -   (lxxxiii) treating nematode infection; or    -   (lxxxiv) treating a disease state, condition, or disorder        mediated by activating transcription factor 4 (ATF4),        in a subject, the method comprising administering to the subject        an effective amount of an ursolic acid salt selected from        ursolic acid diethanolamine salt and ursolic acid morpholine        salt, or a composition comprising the ursolic acid salt.

Various uses as described in the foregoing paragraph are describedrelative to ursolic acid per se in the following references, thedisclosures of which are hereby incorporated by reference herein intheir entirety:

-   1. Wozniak, L., Skapska, S., and Marszalek, K. (2015) Ursolic Acid—A    Pentacyclic Triterpenoid with a Wide Spectrum of Pharmacological    Activities. Molecules 20, 20614-20641-   2. Liu, J. (2005) Oleanolic acid and ursolic acid: research    perspectives. J Ethnopharmacol 100, 92-94-   3. Sultana, N. (2011) Clinically useful anticancer, antitumor, and    antiwrinkle agent, ursolic acid and related derivatives as    medicinally important natural product. J Enzyme Inhib Med Chem 26,    616-642-   4. Liu, J. (1995) Pharmacology of oleanolic acid and ursolic acid. J    Ethnopharmacol 49, 57-68-   5. Shanmugam, M. K., Dai, X., Kumar, A. P., Tan, B. K., Sethi, G.,    and Bishayee, A. (2013) Ursolic acid in cancer prevention and    treatment: molecular targets, pharmacokinetics and clinical studies.    Biochem Pharmacol 85, 1579-1587-   6. Kashyap, D., Tuli, H. S., and Sharma, A. K. (2016) Ursolic acid    (UA): A metabolite with promising therapeutic potential. Life Sci    146, 201-213-   7. Lopez-Hortas, L., Perez-Larran, P., Gonzalez-Munoz, M. J.,    Falque, E., and Dominguez, H. (2018) Recent developments on the    extraction and application of ursolic acid. A review. Food Res Int    103, 130-149-   8. Pironi, A. M., de Araujo, P. R., Fernandes, M. A., Salgado, H. R.    N., and Chorilli, M. (2018) Characteristics, Biological Properties    and Analytical Methods of Ursolic Acid: A Review. Crit Rev Anal Chem    48, 86-93-   9. Cargnin, S. T., and Gnoatto, S. B. (2017) Ursolic acid from apple    pomace and traditional plants: A valuable triterpenoid with    functional properties. Food Chem 220, 477-489-   10. Lee, S. U., Park, S. J., Kwak, H. B., Oh, J., Min, Y. K., and    Kim, S. H. (2008) Anabolic activity of ursolic acid in bone:    Stimulating osteoblast differentiation in vitro and inducing new    bone formation in vivo. Pharmacol Res 58, 290-296-   11. Lu, J., Zheng, Y. L., Wu, D. M., Luo, L., Sun, D. X., and    Shan, Q. (2007) Ursolic acid ameliorates cognition deficits and    attenuates oxidative damage in the brain of senescent mice induced    by D-galactose. Biochem Pharmacol 74, 1078-1090-   12. Wang, X. T., Gong, Y., Zhou, B., Yang, J. J., Cheng, Y.,    Zhao, J. G., and Qi, M. Y. (2018) Ursolic acid ameliorates oxidative    stress, inflammation and fibrosis in diabetic cardiomyopathy rats.    Biomed Pharmacother 97, 1461-1467-   13. Zhou, Y., Li, J. S., Zhang, X., Wu, Y. J., Huang, K., and    Zheng, L. (2010) Ursolic acid inhibits early lesions of diabetic    nephropathy. Int J Mol Med 26, 565-570-   14. Ling, C., Jinping, L., Xia, L., and Renyong, Y. (2013) Ursolic    Acid provides kidney protection in diabetic rats. Curr Ther Res Clin    Exp 75, 59-63-   15. Ding, H., Wang, H., Zhu, L., and Wei, W. (2017) Ursolic Acid    Ameliorates Early Brain Injury After Experimental Traumatic Brain    Injury in Mice by Activating the Nrf2 Pathway. Neurochem Res 42,    337-346-   16. Ma, J. Q., Ding, J., Zhang, L., and Liu, C. M. (2015) Protective    effects of ursolic acid in an experimental model of liver fibrosis    through Nrf2/ARE pathway. Clin Res Hepatol Gastroenterol 39, 188-197-   17. Zhang, T., Su, J., Wang, K., Zhu, T., and Li, X. (2014) Ursolic    acid reduces oxidative stress to alleviate early brain injury    following experimental subarachnoid hemorrhage. Neurosci Lett 579,    12-17-   18. Hwang, T. L., Shen, H. I., Liu, F. C., Tsai, H. I., Wu, Y. C.,    Chang, F. R., and Yu, H. P. (2014) Ursolic acid inhibits superoxide    production in activated neutrophils and attenuates trauma-hemorrhage    shock-induced organ injury in rats. PLoS One 9, e111365-   19. Martinez-Abundis, E., Mendez-Del Villar, M., Perez-Rubio, K. G.,    Zuniga, L. Y., Cortez-Navarrete, M., Ramirez-Rodriguez, A., and    Gonzalez-Ortiz, M. (2016) Novel nutraceutic therapies for the    treatment of metabolic syndrome. World J Diabetes 7, 142-152-   20. Somova, L. O., Nadar, A., Rammanan, P., and Shode, F. O. (2003)    Cardiovascular, antihyperlipidemic and antioxidant effects of    oleanolic and ursolic acids in experimental hypertension.    Phytomedicine 10, 115-121-   21. Somova, L. I., Shode, F. O., Ramnanan, P., and Nadar, A. (2003)    Antihypertensive, antiatherosclerotic and antioxidant activity of    triterpenoids isolated from Olea europaea, subspecies africana    leaves. J Ethnopharmacol 84, 299-305

In some embodiments, the ursolic acid salts described herein are usefulfor, inter alia, the same indications as ursolic acid per se.

As used herein, the term “subject” refers to an animal that is thetarget of administration. In some embodiments, the subject is a human.In other embodiments, the subject is a non-human animal. In someembodiments, the subject is a non-rodent animal. In some embodiments,the subject is a non-human, and/or non-rodent animal. In someembodiments, the subject is a vertebrate and/or an amphibian. In someembodiments, the subject is a human, non-human primate, horse, pig,rabbit, dog, sheep, goat, cow, cat, guinea pig, hamster, ferret, fish,bird, or rodent. In some embodiments, the subject is a human or adomesticated animal. In some embodiments, the subject is a domesticatedanimal, such as a domesticated fish, domesticated crustacean, ordomesticated mollusk. In some embodiments, the domesticated animal ispoultry. In some embodiments, the poultry is selected from chicken,turkey, duck, and goose. In some embodiments, the domesticated animal islivestock. In some embodiments, the livestock animal is selected from apig, cow, horse, goat, bison, and sheep. In some embodiments, thedomestic animal includes rodents. In other embodiments, the domesticanimal excludes some or all rodents. In some embodiments, the domesticanimal excludes mice and rats.

As used herein, the term “treatment” and various forms thereof (e.g.,“treating”) refer to the management (e.g., medical management) of apatient/subject with the intent to cure, ameliorate, stabilize, orforestall a disease, pathological condition, or disorder. The termincludes administering an inventive compound and/or composition to asubject with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect a disease state, condition, ordisorder, the symptoms of the disease state, condition, or disorder orthe predisposition toward the disease state, condition, or disorder. Theterm “treatment” and various forms thereof include the use for aestheticand self-improvement purposes. For example, such uses include, but arenot limited to, the administration of the disclosed compound innutraceuticals, medicinal food, energy bar, energy drink, supplements(such as multivitamins). This term includes active treatment, that is,treatment directed specifically toward the improvement of a disease,pathological condition, or disorder, and also includes causal treatment,that is, treatment directed toward removal of the cause of theassociated disease, pathological condition, or disorder. In addition,this term includes palliative treatment, that is, treatment designed forthe relief of symptoms rather than the curing of the disease,pathological condition, or disorder; preventative treatment, that is,treatment directed to minimizing or partially or completely inhibitingthe development of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder. In variousembodiments, the term covers any treatment of a subject, and includes:(i) impeding the disease from occurring in a subject that can bepredisposed to the disease but has not yet been diagnosed as having it;(ii) inhibiting the disease, i.e., arresting its development; or (iii)relieving the disease, i.e., causing regression of the disease. The termadditionally includes forestalling or impeding the onset, recurrence orintensification of a disease state, condition, or disorder disclosedherein, and ameliorating and/or reducing the occurrence of symptoms of adisease state, condition, or disorder.

Embodiments of the methods of treatment and uses of the inventiveursolic acid salts typically comprise administering an effective amount(e.g., a therapeutically effective amount) of the salt to a subject inneed thereof. As used herein, the term “therapeutically effectiveamount” or “effective amount” refers to an amount that is effective toelicit the desired biological or medical response, including the amountof a compound that, when administered to a subject for treating adisorder, is sufficient to effect such treatment of the disorder. Theeffective amount can vary depending on the compound, the disorder, andits severity, and the age, weight, etc. of the subject to be treated.The effective amount may be in one or more doses (for example, a singledose or multiple doses may be required to achieve the desired treatmentendpoint). An effective amount may be considered to be given in aneffective amount if, in conjunction with one or more other agents, adesirable or beneficial result may be or is achieved. A subject in needof treatment includes a subject that is at risk of needing thetreatment. As used herein, an “at risk” subject is one that is at riskof developing a condition or disorder to be treated. This may be shown,for example, by one or more risk factors, which are measurableparameters that correlate with development of a condition or disorderand are known in the art.

In some embodiments, the subject has been identified to be in need oftreatment for a condition or disorder, or has been diagnosed with acondition or disorder prior to administering an inventive salt to thesubject. In some embodiments, the subject has not been diagnosed with acondition or disorder prior to administering an inventive salt to thesubject. In some embodiments, the subject has not been diagnosed withcancer or diabetes. In other embodiments, the subject may have beendiagnosed with a condition or disorder such as cancer or diabetes.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by theinventive ursolic acid salts, compositions, or methods/uses disclosedherein. For example, “diagnosed with a muscle atrophy disorder” meanshaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition that can bediagnosed or treated by a compound or composition that can promotemuscle health, promote normal muscle function, and/or promote healthyaging muscles. As a further example, “diagnosed with a need forpromoting muscle health” refers to having been subjected to a physicalexamination by a person of skill, for example, a physician, and found tohave a condition characterized by muscle atrophy or other diseasewherein promoting muscle health, promoting normal muscle function,and/or promoting healthy aging muscles would be beneficial to thesubject. Such a diagnosis can be in reference to a disorder, such asmuscle atrophy, and the like, as discussed herein.

As used herein, the phrase “identified to be in need of treatment for acondition or disorder,” or the like, refers to selection of a subjectbased upon need for treatment of the disorder. For example, a subjectcan be identified as having a need for treatment of a disorder (e.g., adisorder related to muscle atrophy) based upon an earlier diagnosis by aperson of skill and thereafter subjected to treatment for the disorder.As another example, subjects intended for consumption (e.g., fish,production livestock, etc.) may be identified to be in need of treatmentfor, e.g., increasing muscle mass, in order to increase the averageamount of meat that can be obtained per animal.

In some embodiments, the inventive ursolic acid salts are administeredin a dosage ranging from about 0.001 to about 500 mg/kg of subject bodyweight (e.g., 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11,0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23,0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35,0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47,0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59,0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71,0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83,0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95,0.96, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4,11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6,12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8,13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0,16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2,17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4,18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6,19.7, 19.8, 19.9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,496, 497, 498, 499, or 500 mg/kg), including any and all ranges andsubranges therein (e.g., 0.001 to 100 mg/kg, 0.01 to 75 mg/kg, 0.05 to20 mg/kg, 0.1 to 10 mg/kg, etc.).

In some embodiments, the inventive ursolic acid salts are administeredin an amount of 1 to 2,000 mg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280,1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400,1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520,1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640,1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760,1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880,1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, or2000 mg), including any and all ranges and subranges therein (e.g., 20to 1900 mg, 50 to 1800 mg, 75 to 1700 mg, 100 to 1600 mg, etc.). Thus,in some embodiments where the ursolic acid salt is administered in acomposition, the composition will contain such amount of the compound.

In some embodiments, the ursolic acid salt is administered in an amountthat is greater than 50, 75, 100, 150, 200, 250, 300, 400, 500, 600,750, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 mg.

EXAMPLES

The invention will now be illustrated, but not limited, by reference tothe specific embodiments described in the following examples.

Sample Preparation˜Ursolic Acid Diethanolamine Salt

Ursolic acid (1.0450 g) and diethanolamine (208.6 mg) were dissolved inTHF:MeOH (10:1) and allowed to stand, then concentrated and dried toyield a white solid, the diethanolamine salt of ursolic acid (965.0 mg).This product was used as the ursolic acid diethanolamine salt in thetesting described below.

Sample Preparation—Ursolic Acid Morpholine Salt

Ursolic acid (1.0715 g) was dissolved in 20 mL of THF. Morpholine (224mg) was added, and stirred for 1.5 hours. 8 mL of hexanes was added andthe homogeneous solution became cloudy. The salt was stirred at roomtemperature for 12 hours. No significant amount of the salt had formed.The solution was concentrated in vacuo, and the stirred residue gave awhite powder that was isolated by filtration and dried in vacuo to givethe morpholine salt of ursolic acid (867.1 mg). This product was used asthe ursolic acid morpholine salt in the testing described below.

Comparative Grip Strength Testing for Ursolic Acid and Salts Thereof

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either standard chow (control) orstandard chow supplemented with either 0.050% ursolic acid, 0.200%ursolic acid, or one of the indicated ursolic acid salts: ursolicacid-ethanolamine salt; ursolic acid-tris(hydroxymethyl)aminomethanesalt, hereinafter referred to as ursolic acid-“tris” salt; ursolicacid-lysine salt; ursolic acid-diethanolamine salt; ursolicacid-morpholine salt; or ursolic acid-piperazine salt. The ursolicacid-diethanolamine salt and ursolic acid-morpholine salt were preparedas described above. Ursolic acid was commercially obtained, and was usedas a starting material to make the other ursolic acid salts according totechniques analogous to those above and known in the art. The doses ofursolic acid salts were molar-matched to either 0.050% ursolic acid(i.e. 0.057% ursolic acid-ethanolamine salt, 0.063% ursolic acid-trissalt, 0.066% ursolic acid-lysine salt, 0.059% ursolic acid-morpholinesalt, and 0.059% ursolic acid-piperazine salt) or to 0.048% ursolic acid(i.e. 0.060% ursolic acid-diethanolamine salt). After mice had consumedthese diets for 6 weeks, in vivo grip strength was measured. The resultsare shown below in Table I. Data are means±SEM from 12 mice per cohort.**P<0.01 by one-way ANOVA with Dunnett's post test.

TABLE I Grip Strength Diet (% Change vs. Control) Control 0.0 ± 1.90.050% Ursolic Acid 7.7 ± 1.9 0.200% Ursolic Acid  9.9 ± 2.2** 0.057%Ursolic Acid-Ethanolamine Salt 2.8 ± 1.6 0.063% Ursolic Acid-Tris Salt5.2 ± 1.6 0.066% Ursolic Acid-Lysine Salt 7.2 ± 2.3 0.060% UrsolicAcid-Diethanolamine Salt  11.7 ± 2.5** 0.059% Ursolic Acid-MorpholineSalt  11.3 ± 2.1** 0.059% Ursolic Acid-Piperazine Salt 3.7 ± 2.5

As evidenced by the data from Table I, not all salts of ursolic acidexhibit statistically significant improvement in strength. Indeed, manymay be less efficacious than the free acid at the same dose. Ursolicacid-diethanolamine salt and ursolic acid-morpholine salt wereunexpectedly more potent and efficacious than native ursolic acid andother ursolic acid salts.

Ursolic Acid-Morpholine Salt—Grip Strength, Body Weight and Composition,and Skeletal Muscle Fiber Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either standard chow (control) orstandard chow supplemented with 0.059% ursolic acid-morpholine salt.After mice had consumed these diets for 7 weeks, in vivo, grip strengthwas measured, mice were weighed, body composition was assessed by NMR,and quadriceps muscles were dissected for histological analysis ofskeletal muscle fiber cross-sectional diameter. The results are shownbelow in Table II. Body weight data, grip strength data, and NMR data(lean mass, fat mass, % lean and % fat) are means±SEM from 14-15 miceper cohort. Muscle fiber diameter data are means±SEM from >4200 musclefibers per diet. *P<0.05; **P<0.01; ***P<0.001

TABLE II Ursolic Acid- Control Morpholine Salt Initial Body Wt (g) 26.1± 0.4 26.3 ± 0.3  Final Grip Strength (g) 165.5 ± 3.3   181.9 ± 2.4***Final Body Wt (g) 35.6 ± 0.8 34.6 ± 1.0  Final Lean Mass (g) 19.6 ± 0.3 20.8 ± 0.3** Final Fat Mass (g) 10.2 ± 0.9 7.8 ± 1.2 Final % Lean 55.3± 1.4 61.0 ± 2.2* Final % Fat 28.0 ± 2.1 21.4 ± 2.9* Muscle fiberdiameter (μm) 39.7 ± 0.1  44.8 ± 0.2***

The data in Table II demonstrate that ursolic acid-morpholine saltstimulated skeletal muscle hypertrophy, increased lean mass anddecreased fat mass.

Ursolic Acid-Morpholine Salt—Skeletal Muscle Fiber Size Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either standard chow (control) orstandard chow supplemented with 0.059% ursolic acid-morpholine salt.After mice had consumed these diets for 7 weeks, quadriceps muscles weredissected, fixed, cross-sectioned, stained with hematoxylin and eosin,and subjected to photomicrography. FIG. 1A is a photomicrograph ofquadriceps skeletal muscle fibers from the control group. FIG. 1B is aphotomicrograph of quadriceps skeletal muscle fibers from the ursolicacid-morpholine salt group. A comparison of FIGS. 1A and 1B shows thatursolic acid-morpholine salt increased skeletal muscle fiber size.

Ursolic Acid-Morpholine Salt—Adipocyte Size Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either standard chow (control) orstandard chow supplemented with 0.059% ursolic acid-morpholine salt.After mice had consumed these diets for 7 weeks, retroperitoneal fatpads were dissected, fixed, cross-sectioned, stained with hematoxylinand eosin, and subjected to photomicrography. FIG. 2A is aphotomicrograph of adipocytes from retroperitoneal fat pads from thecontrol group. FIG. 2B is a photomicrograph of adipocytes fromretroperitoneal fat pads from the ursolic acid-morpholine salt group. Acomparison of FIGS. 2A and 2B shows that ursolic acid-morpholine saltdecreased adipocyte size.

Ursolic Acid-Morpholine Salt—Obesity and Blood Glucose Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either high-fat chow (control) orhigh-fat chow supplemented with 0.059% ursolic acid-morpholine salt.Just prior to starting these diets, mice were weighed and subjected tobody composition analysis by NMR. After mice had consumed the diets for9 weeks, body weight and body composition were re-assessed,retroperitoneal and epididymal fat pads were dissected and weighed, andnon-fasting blood glucose was measured. The results are shown below inTable III. Data are means±SEM from 15 mice per cohort. *P<0.05;**P<0.01.

TABLE III Ursolic Acid- Control Morpholine Salt Initial Body Wt (g) 24.0± 0.5 23.8 ± 0.4  Initial Lean Mass (g) 16.5 ± 0.4 16.7 ± 0.4  InitialFat Mass (g)  2.0 ± 0.2 1.7 ± 0.2 Initial % Lean 68.7 ± 0.8 70.0 ± 0.7 Initial % Fat  8.4 ± 0.9 7.4 ± 0.8 Final Body Wt (g) 41.6 ± 1.0 38.9 ±1.2* Final Lean Mass (g) 20.3 ± 0.6 21.3 ± 0.6  Final Fat Mass (g) 16.5± 0.9 12.8 ± 1.4* Final % Lean 49.0 ± 1.1 55.3 ± 2.4* Final % Fat 39.4 ±1.6 31.8 ± 3.1* Retroperitoneal Fat Pad Wt (mg) 929.4 ± 52.0 731.8 ±86.9* Epididymal Fat Pad Wt (mg) 2257.0 ± 84.6   1804.0 ± 161.5** BloodGlucose (mg/dL) 198.1 ± 6.1  177.7 ± 9.9* 

The data in Table III demonstrate, inter alia, that ursolicacid-morpholine salt reduced obesity in diet-induced obese mice.

Ursolic Acid-Morpholine Salt—Glucose Tolerance Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either high-fat chow (control) orhigh-fat chow supplemented with 0.059% ursolic acid-morpholine salt.After mice had consumed the diets for 8 weeks, mice were fasted for 6hours and then subjected to glucose tolerance testing. Results are shownin FIG. 3, which is a graph depicting glucose tolerance testing results.Data are means±SEM from 15 mice per cohort. As evident from FIG. 3, thearea under the curve (AUC) for mice treated with ursolic acid-morpholinesalt (31,383±1,869) is significantly lower than the AUC for control mice(37,122±1,339; P<0.01), indicating that ursolic acid-morpholine saltsignificantly improved glucose tolerance (i.e. reduced glucoseintolerance).

Ursolic Acid-Diethanolamine Salt—Body Weight and Composition Testing

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either standard chow (control), standardchow supplemented with 0.060% ursolic acid-diethanolamine salt, orstandard chow supplemented with 0.180% ursolic acid-diethanolamine salt.After mice had consumed these diets for 7 weeks, mice were weighed andbody composition was assessed by NMR. Results are shown below in TableIV. Data are means±SEM from 15 mice per cohort. *P<0.05.

TABLE IV 0.060% Ursolic 0.180% Ursolic Acid- Acid- DiethanolamineDiethanolamine Control Salt Salt Initial Body Wt (g) 22.8 ± 0.3 23.0 ±0.3 22.8 ± 0.3 Final Body Wt (g) 31.8 ± 0.9 31.7 ± 0.5 30.7 ± 0.5 FinalLean Mass (g) 19.3 ± 0.3  20.3 ± 0.4* 20.0 ± 0.3 Final Fat Mass (g)  7.1± 1.0  5.3 ± 0.7  4.4 ± 0.6* Final % Lean 61.2 ± 2.0 64.2 ± 1.6  65.5 ±1.4* Final % Fat 21.4 ± 2.5 16.6 ± 2.0  14.1 ± 1.8*

The Table IV data establish that ursolic acid-diethanolamine saltincreased lean mass and decreased fat mass.

Direct Comparison of Ursolic Acid-Morpholine Salt to a Molar-MatchedDose of Ursolic Acid in Diet-Induced Obesity

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either high-fat chow (control), high-fatchow supplemented with 0.050% ursolic acid, or high-fat chowsupplemented with a molar-matched dose of ursolic acid-morpholine salt(0.059%). Just prior to starting these diets, mice were weighed andsubjected to body composition analysis by NMR. After mice had consumedthe diets for 8 weeks, body weight and body composition werere-assessed, retroperitoneal and epididymal fat pads were dissected andweighed, and skeletal muscles (tibialis anterior, gastrocnemius, soleus,quadriceps and triceps) were dissected and weighed. In each mouse, thetotal dissected skeletal muscle weight (combined weight of bilateraltibialis anterior, gastrocnemius, soleus, quadriceps and tricepsmuscles) was normalized to the final body weight. Data are means±SEMfrom 10 mice per cohort. Different letters denote statisticallysignificant differences (P<0.05 by one-way ANOVA with Tukey's posttest). Results are summarized in Table V.

TABLE V 0.059% Ursolic Acid- Control 0.050% Ursolic Acid Morpholine SaltInitial Body Wt (g) 23.4 ± 0.3 ^(a) 23.2 ± 0.3 ^(a) 23.4 ± 0.6 ^(a)Initial Lean Mass (g) 16.0 ± 0.3 ^(a) 15.9 ± 0.4 ^(a) 15.9 ± 0.5 ^(a)Initial Fat Mass (g)  1.6 ± 0.2 ^(a)  1.2 ± 0.1 ^(a)  1.3 ± 0.1 ^(a)Initial % Lean 68.1 ± 0.6 ^(a) 68.5 ± 0.8 ^(a) 68.0 ± 0.7 ^(a) Initial %Fat  6.8 ± 0.8 ^(a)  5.3 ± 0.6 ^(a)  5.6 ± 0.6 ^(a) Final Body Wt (g)42.3 ± 0.7 ^(a) 40.8 ± 0.5 ^(a) 36.6 ± 0.9 ^(b) Final Lean Mass (g) 20.9± 0.3 ^(a) 20.6 ± 0.3 ^(a) 20.9 ± 0.7 ^(a) Final Fat Mass (g) 17.3 ± 0.8^(a) 16.3 ± 0.3 ^(a) 11.6 ± 1.2 ^(b) Final % Lean 49.6 ± 1.0 ^(a) 50.4 ±0.5 ^(a) 57.3 ± 2.1 ^(b) Final % Fat 40.9 ± 1.5 ^(a) 39.8 ± 0.7 ^(a)31.4 ± 2.8 ^(b) Retroperitoneal Fat 1041.0 ± 57.7 ^(a)  986.1 ± 21.8^(a) 709.8 ± 72.2 ^(b) Pad Wt (mg) Epididymal Fat 2449.9 ± 90.0 ^(a) 2208.6 ± 52.0 ^(a)  1713.8 ± 139.8 ^(b) Pad Wt (mg) Skeletal Muscle Wt24.8 ± 0.6 ^(a) 25.5 ± 0.4 ^(a) 29.1 ± 1.0 ^(b) (mg/g Final Body Wt)

Direct Comparison of Ursolic Acid-Morpholine Salt to a Molar-MatchedDose of Ursolic Acid in Obesity-Induced Glucose Intolerance

Weight-matched cohorts of 8-week old male C57BL/6 mice were randomizedto receive ad libitum access to either high-fat chow supplemented with0.050% ursolic acid, or high-fat chow supplemented with a molar-matcheddose of ursolic acid-morpholine salt (0.059%). After mice had consumedthe diets for 7 weeks, mice were fasted for 6 hours and then subjectedto glucose tolerance testing. Results are shown in FIG. 4, which is agraph depicting glucose tolerance testing results. Data are means±SEMfrom 10 mice per cohort. The fasting blood glucose for mice treated withursolic acid-morpholine salt (140.6±7.8 mg/dL) is significantly lowerthan the fasting blood glucose for mice treated with ursolic acid(166.3±3.7; P<0.01). In addition, the area under the curve (AUC) formice treated with ursolic acid-morpholine salt (32,606±2,471) issignificantly lower than the AUC for mice treated with ursolic acid(40,585±1,357; P<0.01), indicating that ursolic acid-morpholine saltsignificantly reduced glucose intolerance relative to a molar-matcheddose of ursolic acid.

Ursolic Acid-Morpholine Salt is More Efficacious than a MaximallyEffective Dose of Ursolic Acid

As an additional side-by-side comparison, testing was undertaken wherebymice were provided ad lib access to high-fat diet containing either0.059% ursolic acid-morpholine salt or a >3-fold higher, maximallyeffective dose of ursolic acid (0.20% w/w; see Kunkel et al., Ursolicacid increases skeletal muscle and brown fat and decreases diet-inducedobesity, glucose intolerance and fatty liver disease, PLOS One 7, (2012)e39332; Kunkel et al., mRNA expression signatures of human skeletalmuscle atrophy identify a natural compound that increases muscle mass,Cell Metabolism 13, (2011) 627-638). Specifically, weight-matchedcohorts of 8-wk-old male C57BL/6 mice were randomized to receive adlibitum access to high-fat diet containing either a maximally effectivedose of ursolic acid (0.20% w/w) or 0.059% (w/w) ursolic acid-morpholinesalt. After 7 weeks on the diets, mice were fasted for 6 hours and werethen subjected to glucose tolerance tests. Results are shown in FIG. 5,which is a graph depicting glucose tolerance testing results. Data aremeans±SEM from 8 mice/diet. The area under the curve (AUC) for ursolicacid-morpholine salt is significantly lower than the AUC for ursolicacid (P<0.05). Relative to the greater than 3-fold higher, maximallyeffective dose of ursolic acid, ursolic acid-morpholine saltsignificantly improved glucose tolerance (FIG. 5) as well as otherparameters including grip strength (P=0.05). These data provide furtherevidence that ursolic acid-morpholine salt is significantly more potentand significantly more efficacious than ursolic acid.

Ursolic Acid-Morpholine Salt is More Efficacious and More Potent thanUrsolic Acid

A direct, side-by-side comparison of ursolic acid and ursolicacid-morpholine salt was performed in an in vitro model of skeletalmuscle. C2C12 myoblasts were cultured and fully differentiated intopost-mitotic skeletal myotubes, then incubated for 48 hours with 10%fetal bovine serum (FBS) plus the indicated concentrations of ursolicacid or ursolic acid-morpholine salt. Myotubes were then harvested forassessment of total cellular protein, which increases as myotubesundergo hypertrophy. (See Dyle et al., Systems-based discovery oftomatidine as a natural small molecule inhibitor of skeletal muscleatrophy, Journal of Biological Chemistry 289, (2014) 14913-14924.) Totalprotein in each sample was normalized to the amount of invehicle-treated myotubes. Each data point represents the mean±SEM of >5samples. *P<0.05 relative to the equivalent dose of ursolic acid.Results are shown in FIG. 6, which is a graph depicting results from thedirect, side-by-side comparison of ursolic acid and ursolicacid-morpholine salt in the cultured C2C12 myotubes in vitro model ofskeletal muscle. As shown, both ursolic acid and ursolic acid-morpholinesalt significantly increased total cellular protein in a dose-dependentmanner, consistent with their capacity to induce myotube hypertrophy.However, ursolic acid-morpholine salt was 3-fold more efficacious and3-fold more potent than ursolic acid.

As used herein, the terms “comprise” (and any form of comprise, such as“comprises” and “comprising”), “have” (and any form of have, such as“has” and “having”), “include” (and any form of include, such as“includes” and “including”), “contain” (and any form contain, such as“contains” and “containing”), and any other grammatical variant thereof,are open-ended linking verbs. As a result, a method or composition that“comprises”, “has”, “includes” or “contains” one or more steps orelements possesses those one or more steps or elements, but is notlimited to possessing only those one or more steps or elements.Likewise, a step of a method or an element of an article that“comprises”, “has”, “includes” or “contains” one or more featurespossesses those one or more features, but is not limited to possessingonly those one or more features.

As used herein, the terms “comprising,” “has,” “including,”“containing,” and other grammatical variants thereof encompass the terms“consisting of” and “consisting essentially of.”

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed compositions or methods.

Where one or more ranges are referred to throughout this specification,each range is intended to be a shorthand format for presentinginformation, where the range is understood to encompass each discretepoint within the range as if the same were fully set forth herein.

While several aspects and embodiments of the present invention have beendescribed and depicted herein, alternative aspects and embodiments maybe affected by those skilled in the art to accomplish the sameobjectives. Accordingly, this disclosure and the appended claims areintended to cover all such further and alternative aspects andembodiments as fall within the true spirit and scope of the invention.

1. An ursolic acid salt selected from ursolic acid diethanolamine saltand ursolic acid morpholine salt.
 2. The ursolic acid salt according toclaim 1, wherein the salt is ursolic acid diethanolamine salt of formula


3. The ursolic acid salt according to claim 1, wherein the salt isursolic acid morpholine salt of formula


4. A composition comprising a salt according to claim 2, and a furtheragent.
 5. The composition according to claim 4, comprising 25 to 1,500mg of the salt.
 6. The composition according to claim 4, comprising 50to 1,000 mg of the salt.
 7. A composition comprising a salt according toclaim 3, and a further agent.
 8. The composition according to claim 7,comprising 25 to 1,500 mg of the salt.
 9. The composition according toclaim 7, comprising 50 to 1,000 mg of the salt.
 10. The compositionaccording to claim 4, wherein the further agent comprises aphysiologically acceptable carrier.
 11. The composition according toclaim 7, wherein the further agent comprises a physiologicallyacceptable carrier.
 12. The composition according to claim 4, whereinthe composition is a pharmaceutical composition, nutraceuticalcomposition, food composition, or dietary supplement.
 13. Thecomposition according to claim 7, wherein the composition is apharmaceutical composition, nutraceutical composition, food composition,or dietary supplement.
 14. The composition according to claim 12,comprising 25 to 1,500 mg of the salt.
 15. The composition according toclaim 13, comprising 25 to 1,500 mg of the salt.
 16. A method for: (i)increasing skeletal muscle mass; (ii) treating skeletal muscle atrophy;(iii) treating sarcopenia; (iv) treating cachexia; (v) increasingstrength; (vi) treating weakness; (vii) increasing exercise capacity;(viii) treating fatigue; (ix) promoting muscle growth; (x) promotingnormal muscle function; (xi) improving muscle function; (xii) promotingmuscle health; (xiii) promoting healthy aging in muscles; (xiv)increasing energy expenditure; (xv) increasing the ratio of skeletalmuscle to fat; (xvi) reducing fat; (xvii) treating obesity; (xviii)treating diabetes; (xix) lowering blood glucose; (xx) treatingpre-diabetes; (xxi) treating metabolic syndrome; (xxii) treating insulinresistance; (xxiii) reducing plasma cholesterol; (xxiv) treatinghypercholesterolemia; (xxv) reducing plasma triglycerides; (xxvi)treating hypertriglyceridemia; (xxvii) promoting healthy metabolism;(xxviii) promoting metabolic health; (xxix) treating hypertension; (xxx)treating atherosclerosis (xxxi) treating myocardial ischemia; (xxxii)treating myocardial infarction; (xxxiii) treating cardiomyopathy;(xxxiv) treating cardiac arrhythmia; (xxxv) treating non-alcoholic fattyliver disease (NAFLD); (xxxvi) treating liver fibrosis; (xxxvii)treating liver injury; (xxxviii) treating lung injury; (xxxix) treatinggastric ulcers; (xl) treating nephropathy; (xli) promoting boneformation; (xlii) promoting normal bone structure; (xliii) promotingbone health; (xliv) treating osteoporosis; (xlv) treating cerebralischemia; (xlvi) treating cerebral hemorrhage; (xlvii) treating stroke;(xlviii) treating traumatic brain injury; (xlix) treating dementia; (l)treating Alzheimer's disease; (li) treating memory loss; (lii) treatingcognitive dysfunction; (liii) promoting normal cognitive function; (liv)treating anxiety; (lv) treating depression; (lvi) reducing inflammation;(lvii) treating arthritis; (lviii) treating skin ulcers; (lix) treatingskin wounds; (lx) promoting wound healing; (lxi) treating skin dryness;(lxii) treating skin roughness; (lxiii) treating skin scarring; (lxiv)treating skin wrinkles; (lxv) reducing unwanted effects of aging; (lxvi)treating cancer; (lxvii) reducing tumor growth; (lxviii) treating tumormetastasis; (lxix) treating tumor angiogenesis; (lxx) increasing tumorcell apoptosis; (lxxi) decreasing protein oxidation; (lxxii) decreasinglipid oxidation; (lxxiii) decreasing DNA oxidation; (lxxiv) decreasingRNA oxidation; (lxxv) decreasing oxidation of cellular molecules;(lxxvi) decreasing DNA damage; (lxxvii) treating bacterial infection;(lxxviii) reducing bacterial growth; (lxxix) treating fungal infection;(lxxx) reducing fungal growth; (lxxxi) treating viral infection;(lxxxii) treating protozoal infection; (lxxxiii) treating nematodeinfection; or (lxxxiv) treating a disease state, condition, or disordermediated by activating transcription factor 4 (ATF4), in a subject, themethod comprising administering to the subject an effective amount of asalt according to claim
 1. 17. The method according to claim 16, whereinthe salt is ursolic acid morpholine salt.
 18. The method according toclaim 16, comprising administering to the subject a composition thatcomprises 25 to 1,500 mg of the salt.
 19. The method according to claim18, wherein the subject is a human, non-human primate, horse, pig,rabbit, dog, sheep, goat, cow, cat, guinea pig, hamster, ferret, fish,bird, or rodent.
 20. The method according to claim 16, wherein thesubject has not been diagnosed with diabetes or cancer.